Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have demonstrated that the regions near telomeres are highly sensitive to DNA double-strand breaks (DSBs), in that DSBs induced with I-SceI endonuclease near telomeres are much more likely to result in large deletions, gross chromosome rearrangements (GCRs), and chromosome instability than I-SceI-induced DSBs at other locations. Importantly, the rearrangements caused by DSBs near telomeres are the same rearrangements commonly found in human cancer cells, leading us to propose that DSBs near telomeres are an important mechanism in carcinogenesis. We have also shown that the chromosome instability caused by DSBs near telomeres can be prevented by the addition of a new telomere at the site of the DSB, a process called chromosome healing. Chromosome healing is rarely observed at DSBs at other locations, and therefore we have proposed that chromosome healing is an important mechanism for preventing chromosome instability due to DSBs near telomeres. This proposal will investigate the mechanism responsible for the sensitivity of telomeric regions to DSBs and the mechanism of regulation of chromosome healing. These studies will address the hypothesis that cis-acting telomeric proteins directly inhibit DSB repair and promote chromosome healing, consistent with evidence that the telomeric protein TRF2 inhibits the ATM and MRE11 proteins involved in the cellular response to DSBs. In Aim 1A we will characterize the DNA repair defect in telomeric regions by determining which DNA repair proteins co-localize with the I-SceI-induced DSB. This will involve cell lines in which the location of the DSB is marked with green fluorescent protein (GFP) by inserting 256 copies of a LacO operon adjacent to the I-SceI site, and expression of LacI-GFP fusion protein, which binds the LacO operon. In Aim 1B we will use cell clones containing a GFP gene and an I-SceI site adjacent to a telomere to monitor how knockdown of telomeric proteins or DSB repair proteins affects the frequency of large deletions. In Aim 1C we will characterize the DSB repair proteins involved in the formation of chromosome aberrations using cell clones that contain a GFP gene activated by intrachromosomal rearrangements, and a DsRed gene activated by interchromosomal rearrangemetns. In Aim 2A we will use a novel real-time quantitative PCR assay for chromosome healing to monitor how knockdown of telomeric proteins or ATM affects the frequency of chromosome healing in isogenic cell clones generated by moving a telomere to a location adjacent to the I-SceI site using Cre/LoxP-mediated recombination. In Aim 2B we will compare the appearance of PIF1 helicase, a protein known to inhibit chromosome healing in yeast, at subtelomeric and interstitial DSBs with and without knockdown of TRF2 or ATM, using the same cell clones containing the DSBs marked with the LacI-GFP fusion protein used in Aim 1A. PUBLIC HEALTH RELEVANCE: Chromosome instability is an important factor in promoting the multiple genetic changes leading to cancer. We have demonstrated that one mechanism for chromosome instability in human cancer cells involves the sudden loss of telomeres, the caps that protect the ends of chromosomes and prevent chromosome fusion. This proposal will investigate the mechanisms for two factors that we have shown are important in this process. The first is the sensitivity of regions near telomeres to double-strand breaks, which we believe is an important factor in the increased rate of telomere loss in cancer cells. The second is restoration of lost telomeres, called chromosome healing, which we have shown can prevent chromosome instability due to telomere loss. Understanding the mechanisms of telomere loss and chromosome healing in human cancer cells may lead to new approaches for the limiting the chromosome instability responsible for cancer cell progression and resistance to anti-cancer therapies.